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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
there were minor deviations not affecting the validity of the study
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
DETAILS ON PREPARATION
As the solubility of the test item in the test medium was below 100 mg/L, the test solution was prepared as follows. The load of 781 mg/L (corresponding to a volume of 0.884 mL/L based on a density of 0.883 g/mL) was added to the corresponding amount of nutrient medium (demineralised water enriched with minerals but without algae) and homogenised for 30 minutes at 30°C in an ultrasonic bath. Afterwards, the solution was stirred for 30 minutes on a magnetic stirrer. Then, the solution was homogenised again using ultrasonic for 15 min and afterwards stirred for 15 minutes. The resulting clouded solution was filtrated through 0.45 µm nylon filters. An aliquot was examined for colloidal particles by testing the solution with a laser for the Tyndall effect.
This solution was stated as the 100 % saturated solution. The lower treatments were prepared by dilution of this solution. According to the information of the sponsor, stability was not given over a period of more than 24 hours. In the alga test, a medium renewal every 24h of the test solution to ensure exposure to the parent substance is not possible. Therefore, it was expected that exposure to the degradation products occurred after 24h.
The study was performed using five concentrations ranging from 78.1 to 781 mg/L nominal concentration resp. 10 – 100 % saturation. Incubation time was 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the solutions at 440 nm every 24 hours with a spectral photometer. The cell density of the cultures was calculated based on the correlation curve between the adsorption and the cell density of the cultures determined by microscope counts. Growth rate µ, area under the growth curve (AUC ) and the yield were determined from the cell densities at the respective observation times.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Unicellular freshwater green alga.
Genus, Species Desmodesmus subspicatus
Strain CHODAT
Family Scenedesmacea
Order Sphaeropleales

Origin and Culture:
The culture of Desmodesmus subspicatus was obtained in Oct. 2011 by MBM Science-bridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 8 °C. From the stock culture, a permanent culture was prepared.

Seven days before the start of the test, an aliquot of the stock culture containing a few cells was brought into pre-culture medium and incubated for 72 hours. The resulting culture is growing exponentially.
Before usage, the culture was checked on the absence of cell aggregates.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 – 25 °C
Nominal and measured concentrations:
Nominal concentrations: 10 / 18 / 32 / 56 / 100 % saturation (loading rate of 781 mg/L)
Limit of solubility = approx. 2.8 mg/L, based on the geometric mean of the measured DOC concentration in the fresh prepared solutions
Details on test conditions:
Date: 22. – 25. Feb. 2013
Treatments tested: 10 / 18 / 32 / 56 / 100 % saturation
Number of replicates: six replicates for the control
three replicates for each treatment
Vessels: glass flasks 50 mL
Duration: 72 hours
Temperature: 24 – 25 °C
Lighting: 5800 Lux
Control: deionised water with nutrient medium and alga
Treatments: test solution with nutrient medium and alga

For each treatment, 200 mL of the respective test item solution was mixed with the necessary amount of algal pre-culture (0.77 mL) to achieve a cell concentration of approx. 2 *103 cells/mL. In this mixture, the pH-value was measured.
For the control, nutrient medium was used instead of test item solution. The test vessels were completely filled with the respective test solution and incubated for 72 hours, shaken on an orbital shaker. Before the start of incubation and every 24 hours, the cell number was calculated based on the determination of the absorption at 440 nm. After the test, the pH value in treatments and control was measured again.
At the end of the test, the treatments were examined microscopically in order to assess the appearance of the alga and detect abnormalities (e.g. caused by the exposure to the test item).
The content of the test item in the test vessels was measured at the start and every 24 hours during the test throug DOC measurements.
Due to a writing error in the study plan, the test concentration was 781 mg/L instead of 100 mg/L. As both concentrations are far above the limit of solubility of the test item and the saturated solution was tested this was stated as uncritical.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 781 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 2.6 other: mg/L (approximately equal to the solubility limit)
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 3.1 other: mg/L (approx. the solubility limit)
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
ca. 1.6 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
For the endpoints µ, AUC and Yield, the differences between the nominal treatment 1.12 mg/L (32 % saturation) and the control can be considered as not significant (level of significance: 97.5 %) as the calculated t-values were smaller than the limit of significance. Therefore, the nominal concentration 1.12 mg/L (32 % saturation) is stated as NOEC for these endpoints.


For the endpoints µ, AUC and Yield, the differences between the nominal treatment 1.96 mg/L (56 % saturation) and the control must be considered as significant (level of signifi-cance: 97.5 %), the nominal concentration 1.96 mg/L (56 % saturation) can be stated as LOEC for these endpoints.
Results with reference substance (positive control):
The EC50s of potassium dichromate were determined in a separate GLP study. For the estimation of the EC50s of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the normal range of the laboratory (72h ErC50 between 0.60 – 1.03 mg/L).
Reported statistics and error estimates:
The estimation of the EC50s of the test item was accomplished using the software OriginTM.
The data were evaluated using linear fit on a probability-logarithmic scale.

The cell numbers were determined by photometric measurement of optical density. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Nominal Concentration in % saturation

Parameter

Cell Number/mL

 

 

0 h

24 h

48 h

72 h

0

Mean

2206

26840

144493

555544

0

SD

0

2483

15999

13211

10

Mean

2206

29413

147802

549662

10

SD

0

4176

9616

21341

18

Mean

2206

36767

138610

533852

18

SD

0

3184

10483

7954

32

Mean

2206

27207

136037

542308

32

SD

0

1685

7091

8568

56

Mean

2206

35296

115080

455539

56

SD

0

5515

24399

51405

100

Mean

2206

20589

79048

276485

100

SD

0

8279

6463

11690

Inhibition:

The following mean inhibition values were calculated for the Growth Rate µ, the Area under the Curve AUC, and the Yield:

Nom. Concentration in % saturation

Measured Concentration in mg/L

% Inhibition

Growth Rate µ

Area under the Curve AUC

Yield

0

--

-

-

-

10

--

0.20

-0.66

1.06

18

--

0.72

1.53

3.92

32

1.12

0.43

3.32

2.39

56

1.96

3.66

16.00

18.07

100

2.77

12.63

47.62

50.43

Validity criteria fulfilled:
yes
Remarks:
The cell concentration in the control increased by a factor of 252. Mean coefficient of variation of daily growth rates was 32% and coefficient of variation of average growth rate during the test was < 1%. The change in the pH of the control was 0.1 unit.
Conclusions:
72h ErC50 > limit of solubility (approx. 2.7 mg/L in the test media) with a loading rate of 781 mg/L
72h ErC10 = 2.6 mg/L (approx. the limit of solubility)

According to the available information on stability, hydrolysis of the substance is expected between 24h and 48h in water. As for the algal test renewal of the test medium is not possible (as done for the fish and daphnid tests), it is likely that the toxicity of the degradation products is observed here, rather than the toxicity of the parent substance PHOSPHOROUS ACID, TRI-C12-14-ALKYL ESTERS.
Executive summary:

The study was performed using five concentrations ranging from 10 – 100 % saturation, using a loading rate of 781 mg/L.

Due to the lack of suitable analytical method, the DOC concentrations were measured in solutions and were used to determine the test item concentration during the test. The limit of solubility of the test item in the algal test media was estimated to be 2.7 mg/L.

The two highest concentrations showed significant toxicity and the following results were determined:

72h ErC50 > limit of solubility (approx. 2.7 mg/L in the test media) with a loading rate of 781 mg/L

72h ErC10 = 2.6 mg/L (approx. the limit of solubility)

According to the available information on the substance stability, degradation is expected in water after 24h. As for the algal test renewal of the medium is not possible, it is expected that the observed toxicity is related to the degradation products (and in particular dodecan-1-ol) rather than the parent substance.

Description of key information

The toxicity of the test item to the freshwater green algae Desmodesmus subspicatus was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 201.

The 72h ErC50 > limit of solubility (approx. 2.7 mg/L in the test media) with a loading rate of 781 mg/L

The 72h ErC10 = 2.6 mg/L (approx. the limit of solubility)

According to the available information on the substance stability, degradation is expected in water after 24h. As for the algal test renewal of the medium is not possible, it is expected that the observed toxicity is related to the degradation products (and in particular dodecan-1-ol) rather than the parent substance.

Key value for chemical safety assessment

Additional information

The toxicity of the test item to freshwater green algae Desmodesmus subspicatus was investigated in one GLP-compliant study performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.